Additional Data File 1
Supporting Materials and Methods
Design and production of microarray
Design and production of guinea pig microarray, microarray processing for various samples and data
analysis was carried out at Genotypic Technology Pvt. Ltd, Bangalore. Development of 44K array is based
on a 244K microarray, which was initially designed to contain ~60-mer oligonucleotide probes from multiple
species. The 244K array comprised of a total number of 2,43,504 features including 2,13,039 probes from
different mammalian species, 2105 Agilent controls and 28,360 blank spots. The number of features from
various mammals that have been used for designing the 244K multi-species array is detailed in Supporting
Table S1. All the oligonucleotides were designed and synthesized in situ as per the standard algorithms and
methodologies used by Agilent Technologies for 60- mer in situ oligonucleotide DNA microarray.
RNA and Genomic DNA isolation
RNA and genomic DNA used for the design and development of guinea pig microarray were isolated
from various tissues such as lung, liver, spleen, brain, muscle, kidney and bone marrow of 6-8 weeks old
guinea pigs. Genomic DNA was extracted from guinea pig tissues by using DNeasy Blood and Tissue Kit as
per the manufacturer’s instructions (Qiagen). Total RNA was isolated from various tissues by using Agilent
Total RNA Isolation Mini kit as per the manufacturer’s instructions (Agilent Technologies)
DNA and RNA Quality Control
Quality and quantity of genomic DNA were determined by using agarose gel electrophoresis and
Agilent Nanodrop spectrophotometer (Agilent Technologies). Intact genomic DNA with A260/280 ≥ 1.8 and
A260/230 ≥ 1.0 was used for microarray hybridization experiments. RNA concentration and purity was also
determined by using the Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies) and the integrity
of RNA was verified on an Agilent 2100 Bioanalyzer by using the RNA 6000 Nano LabChip (Agilent
Technologies). RNA samples with an rRNA 28S/18S ratio ≥ 1.5, rRNA contribution ≥ 30%, RNA integrity
number (RIN) ≥ 6, A260/280 > 1.8 and A260/230 ≥ 1.3 were used for the microarray experiments.
RNA and DNA labeling and microarray hybridization
Total RNA isolated from various guinea pig tissues (lung, liver, spleen, brain, muscle, kidney and
bone marrow) was amplified by using the Agilent Low RNA Input Fluorescent Linear Amplification Kit and
labelled with Cy3 CTP using Agilent Quick Amp Kit PLUS. RNA was then reverse transcribed to double
stranded cDNA by using oligo dT primers. The double stranded cDNA was then used as template for cRNA
generation by in vitro transcription. Labeled cRNA was purified and its quality was assessed for yield and
specific activity by using Agilent Nanodrop spectrophotometer (Agilent Technologies).
Genomic DNA was labeled with Cy5 dUTP by using Agilent Genomic DNA Labeling Kit PLUS.
Briefly, 1.5g of genomic DNA was digested at 37°C with restriction enzymes Alu I and Rsa I and labeled
with dUTP dyes. The labeled RNA and DNA were then purified and assessed for yields and specific activity.
Cy3 labelled cRNA and Cy5 labelled genomic DNA showed a specific activity of 17.57 pmol dye/g cRNA
and 30.35 pmol dye/g DNA, respectively.
The 244K microarray was then hybridized with Cy3 labelled cRNA produced from pooled RNA
obtained from various guinea pig tissues and Cy5 labelled genomic DNA by using standard Agilent in situ
Hybridization kit protocol. Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours.
The hybridized slides were washed by using Agilent Gene Expression wash buffers and scanned by using
the Agilent Microarray Scanner G Model G2565BA at 5m resolution. Similarly, 5 g of Cy5 labeled DNA
was fragmented by using Comparative Genomic Hybridization kit of Agilent and hybridized on the same
array for 40 hours at 65º C, washed and scanned as described earlier. Data was extracted from the images
by using Feature Extraction software (v9.5) from Agilent.
Based on the intensities observed in the above hybridization experiment, the probes showing signal
intensity twice or more than twice the background signal intensity were selected and a 44K array was
designed. As described above, all the oligonucleotides were designed and synthesized in situ as per the
standard algorithms and methodologies used by Agilent Technologies.
Annotation of probes for 44K microarray
The guinea pig 44K microarray comprised of a total number of 45220 features including 29846 valid
features from different mammalian species, 1132 probes for guinea pig transcripts and 12825 probes for
guinea pig ESTs, 1264 Agilent positive controls and 153 Agilent negative controls. The % representation of a
particular species is calculated with respect to the total number of probes in the array. Upon sequence
analysis, 42,511 probes were found to be unique. Of the 42,511 unique probes, 20,574 represented
Reference Sequences (REFSEQ Database), 86 probes corresponded to Agilent proprietary transcript
database, 18,914 probes represented EST database and 2,284 probes represented transcripts unique to
ENSEMBLE and TIGR database. All the guinea pig specific probes were re-annotated by blast based
homology to the transcript sequence database of guinea pig from NCBI (as available in Sep 2011), with a
criterion of best hit of more than 75% identity over a minimum 30 base pair alignment length. 2971 of the
guinea pig specific probes were found to correspond to 344 unique genes of guinea pig.
Further, for biological interpretation, homolog or gene ontology annotation was also obtained for all
the probes by blast based homology to reference sequence database of Human, Mouse and Rat. The
homolog annotation for the probes is based on the criteria of best hit of more than 75% identity over a
minimum 30 base pair alignment length in any of the three organisms, human, mouse or Rat. Annotated
genes that exhibited significant up or down regulation (4190 genes) were included for functional analysis and
are listed in Additional Data File 3. Functional classification of differentially regulated genes was carried out
by using GeneSpringGX software and Genotypic Biointerpreter Biological Analysis software (Genotypic
Technology Pvt. Ltd.). The microarray data reported here have been submitted at NCBI’s Gene Expression
Omnibus [GEO Accession number: GSE32447].
Supporting Tables
Supporting Table S1. Probe distribution in 244K GPOM: The table depicts the number of features
derived from various mammalian species that have been used for designing the 244K GPOM.
Organism
Sequence data source
Number of Probes in 244K Array
Human (Homo Sapiens)
Agilent Catalogue Arrays
36,866
Mouse (Mus musculus)
Agilent Catalogue Arrays
40,896
Rat (Rattus norvegicus)
Agilent Catalogue Arrays
39,381
Agilent Catalogue Arrays
20,217
Dog (Canis familiaris)
Agilent Catalogue Arrays
42,034
Horse (Equus caballus)
NCBI, mRNA sequences
1120
Cat (Felis catus)
NCBI, mRNA sequences
637
Sheep (Ovis aries)
NCBI, mRNA sequences
1356
Pig (Sus scrofa)
NCBI, mRNA sequences
16,003
NCBI, mRNA sequences
1132
NCBI, mRNA sequences
84
NCBI, mRNA sequences
849
NCBI, mRNA sequences
224
NCBI, mRNA sequences
12,240
Total Number of probes
2,13,039
Rhesus Monkey (Macaca
mulatta)
Guinea pig (Cavea
porcellus)
Chinchilla lanigera
Chimpanzee (Pan
troglodyte)
Gray tailed opussum
(Monodelphis domestica)
Cattle (Bos Taurus)
Supporting Table S2. Quantitative real time RT-PCR for microarray validation. The table depicts the primer
sequences employed for real time RT-PCR of C3AR1, C4BPA, CAMP, CCL5, IFN- and 18S rRNA genes and the
gene expression pattern observed with respect to that observed in case of microarray.
Primer
Name
C3AR1
C4BPA
CAMP
CCL5
IFN
18S
Primer Sequence
Primer
Length
(bases)
Amplicon
size
Expression Match with
Tm (º C)
(base
profile
Microarray
pairs)
60
143
Down
Match
60
F
TCTGTATCTCCCTTATTTCACT
22
R
CCTAAGTGCCCATCAACAGA
20
F
GCCTGGATGCTTTGCTGTC
19
60
R
CAAATATACGAGGCACAGATG
21
60
F
CGCGAGTACGGGCAGATC
18
60
R
GGCAAGAACACACTAGGTAG
20
60
F
CTGGCCCACTGCTTAGCAAT
20
60
R
CCTTGCTTCTTTGCCTTGAAA
21
60
F
ACAAGGTGCAGGCTTTCAAAA
21
60
R
TTGGCGCTGGACATGCT
17
54
F
TGCATGGCCGTTCTTAGTTG
20
60
162
Up
Match
163
Down
Match
62
Down
Match
65
Down*
Match
76
R
23
For normalization
68
AGTTAGCATGCCAGAGTCTCGTT
* While one of the samples showed mild down regulation other showed no change by real time RTPCR. By
microarray overall expression for IFN showed mild down regulation.
References: